Binding of Fibrin Monomer and Heparin to Thrombin in a Ternary Complex Alters the Environment of the Thrombin Catalytic Site, Reduces Affinity for Hirudin, and Inhibits Cleavage of Fibrinogen

Abstract

The kinetics of transport of L-lactate, pyruvate, ketone bodies, and other monocarboxylates into isolated hepatocytes from starved rats were measured at 25 degrees C using the intracellular pH-sensitive dye, 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein, to detect the associated proton influx. Transport kinetics were similar, but not identical, to those determined using the same technique for the monocarboxylate transporter (MCT) of Ehrlich Lettré tumor cells (MCT1) (Carpenter, L., and Halestrap, A. P. (1994) Biochem. J. 304, 751-760). Km values for L-lactate (4.7 mM), D-lactate (27 mM), D,L-2-hydroxybutyrate (3.3 mM), L-3-hydroxybutyrate (12.7 mM), and acetoacetate (6.1 mM) were very similar in both cell types, whereas in hepatocytes the Km values were higher than MCT1 for pyruvate (1.3 mM, cf. 0.72 mM), D-3-hydroxybutyrate (24.7 mM, cf. 10.1 mM), D-2-chloropropionate (1.3 mM, cf. 0.8 mM), 4-hydroxybutyrate (18.1 mM, cf. 7.7 mM), and acetate (5.4 mM, cf. 3.7 mM). In contrast, the hepatocyte carrier had lower Km values than MCT1 for glycolate, chloroacetate, dichloroacetate, and 2-hydroxy-2-methylpropionate. Differences in stereoselectivity were also detected; both carriers showed a lower Km for L-lactate than D-lactate, while hepatocyte MCT exhibited a lower Km for D- than L-2-chloropropionate and for L- than D-3-hydroxybutyrate; this is not the case for MCT1. A range of inhibitors of MCT1, including alpha-cyanocinnamate derivatives, phloretin, and niflumic acid, inhibited hepatocyte MCT with K0.5 values significantly higher than for tumor cell MCT1, while stilbene disulfonate derivatives and p-chloromercuribenzene sulfonate had similar K0.5 values in both cell types. The branched chain ketoacids alpha-ketoisocaproate and alpha-ketoisovalerate were also potent inhibitors of hepatocyte MCT with K0.5 values of 270 and 340 microM, respectively. The activation energy of L-lactate transport into hepatocytes was 58 kJ mol-1, and measured rates of transport at 37 degrees C were considerably greater than those required for maximal rates of gluconeogenesis. The properties of the hepatocyte monocarboxylate transporter are consistent with the presence of a distinct isoform of MCT in liver cells as suggested by the cloning and sequencing of MCT2 from hamster liver (Garcia, C. K., Brown, M. S., Pathak, R. K., and Goldstein, J. L. (1995) J. Biol. Chem. 270, 1843-1849).

Crystal structure of the complex between thrombin and the central “E” region of fibrin

“…Therefore, this fragment most probably mimics the corresponding region in fibrin and the interactions described here would reflect those between fibrin and thrombin, if the latter interacts with fibrinogen in a different manner. In this connection, it was reported that thrombin, in a ternary complex with fibrin and heparin, preserves its catalytic activity toward fibrinogen (50,51), suggesting that its active site cleft remains unoccupied. This is in agreement with the structure of our complex in which the active site and the SЈ groove of thrombin appear to be accessible to a substrate (Fig.…”

Binding of Exosite Ligands to Human Thrombin

“…Simultaneous binding of fibrin monomer or polymer to exosite I and high molecular weight heparin to exosite II in a ternary complex with thrombin was facilitated by binding interactions between fibrin and heparin, resulting in a respective 6 -40-fold enhanced affinity of polymer and monomer for the thrombinheparin complex, compared with free thrombin (34,57). However, these substantial increases in affinity are not attributed to allosteric inter-exosite linkage but are the consequence of additional heparin-fibrin interactions.…”

“…Exosite II binds heparin and other glycosaminoglycans (2, 12, 13), prothrombin activation fragment 2 (F2) (14), the chondroitin sulfate moiety of thrombomodulin (15, 16), the leech peptide hemadin (17), and an exosite II-specific human monoclonal antibody (18). Factors V (19 -22), Va (21,22), and VIII (19), platelet glycoprotein Ib␣ (23-25), and the snake venom protein bothrojaracin (26) have been reported to interact with both exosites I and II.Binding of exosite ligands to thrombin is correlated with significant changes in the kinetics of hydrolysis of peptide ester and peptide p-nitroanilide substrates (3,7,9,18,(27)(28)(29)(30)(31) in addition to profound effects on specificity and reactivity toward its natural macromolecular substrates and inhibitors (10,11,15,(32)(33)(34)(35)(36). These studies indicate that exosite binding of allosteric effectors is coupled to conformational changes affecting the S1-S3 substrate specificity subsites in the thrombin catalytic site (37-39).…”

“…These studies indicate that exosite binding of allosteric effectors is coupled to conformational changes affecting the S1-S3 substrate specificity subsites in the thrombin catalytic site (37)(38)(39). Binding studies of F2, thrombomodulin, fibrin, and heparin with various active site-labeled thrombin derivatives in which the S1-S4 subsites were occupied (16,34,40), and studies of the effect of exosite ligand binding on the hydrolysis of tripeptide p-nitroanilide substrates suggest that structurally different ligands produce ligand-specific changes in the catalytic site. Extreme allosteric linkage between exosites I and II (30,31,41) has been reported to prevent simultaneous occupation of exosites I and II (30,41), whereas other studies provide contrasting evidence for binary and ternary complex formation with similar affinities among thrombin and exosite I and II ligands (18).…”

“…Binding of exosite ligands to thrombin is correlated with significant changes in the kinetics of hydrolysis of peptide ester and peptide p-nitroanilide substrates (3,7,9,18,(27)(28)(29)(30)(31) in addition to profound effects on specificity and reactivity toward its natural macromolecular substrates and inhibitors (10,11,15,(32)(33)(34)(35)(36). These studies indicate that exosite binding of allosteric effectors is coupled to conformational changes affecting the S1-S3 substrate specificity subsites in the thrombin catalytic site (37)(38)(39).…”